Genetic variation and cryptic lineage among the sergestid shrimp Acetes americanus (Decapoda)

The taxonomic status of the sergestid shrimp, Acetes americanus, has been questioned for several decades. No specific study has been performed thus far to resolve the incongruences. This species has a wide geographical range in the western Atlantic and is represented by two formally accepted subspecies: Acetes americanus carolinae, distributed in North America, and Acetes americanus americanus, present in South America. However, there are regions where the coexistence of both subspecies has been reported, such as Central America. This study aimed to genetically compare specimens of A. a. americanus collected in South America with A. a. carolinae sampled in North America to check for possible differences and the existence of more than one subspecies of A. americanus on the Brazilian coast. Based on the sequences of two informative markers, the cytochrome oxidase I region (COI) and 16S rRNA, phylogenetic reconstruction demonstrated well-defined clades with high support values, reinforcing the idea that A. a. americanus is genetically different from A. a. carolinae. Our hypothesis was corroborated as the specimens collected in Brazil were divided into two distinct lineages: the first composed of A. a. americanus sensu stricto (Brazil 1) and the second by Acetes americanus (Brazil 2). The three groups evidenced in the haplotype network were the same as those observed in the phylogenetic tree. The morphometric character (height/length of the thelycum) was effective in distinguishing A. a. Brazil 1 from A. a. carolinae. However, more detailed and conclusive studies comprising other characteristics to propose and describe a possible new entity are necessary. To the best of our knowledge, for the first time, the results of this study provide some insights into the taxonomic status of the sergestid shrimp A. americanus in the western Atlantic.

In this study, we compared the A. a. americanus specimens collected from South America with those of A. a. carolinae sampled in North America using COI and 16S rRNA markers to test the genetic validity of both subspecies and the possible existence of other entities distributed along the western Atlantic.

DNA extraction and amplification
DNA extraction from the muscular abdominal tissue was performed following Mantelatto, Robles & Felder (2007); Mantelatto et al. (2009a); Mantelatto et al. (2009b) and Pileggi & Mantelatto (2010), with specific modifications (Carvalho-Batista et al., 2014). The regions of interest were amplified by the polymerase chain reactions (PCR) using the primers described in Table S2. Primers specific for this region were designed due to the difficulty in the amplification of COI (see Mantelatto et al., 2016 for details) (Table S2). PCR reactions were performed with a total volume of 25 µL, containing 5 µL of betaine (5M) (Acros Organics), 4 µL of dNTPs (5 mM), 3 µl of MgCl 2 (25 mM), 3 µl of 10X Taq buffer with KCl (Thermo Scientific), 2 µl of 1% bovine albumin (Sigma), 1 µl of each primer (10 µM), 1 µl of resuspended DNA (50 ng/ ml), and 0.5 µl of recombinant Taq DNA Polymerase (Thermo Scientific). The missing volume (25 µl) was filled with ultrapure water. PCR was performed using an Applied Biosystems c Veriti 96 well thermal cycler. PCR steps comprised an initial denaturation period of 3 min at 95 • C, followed by 40 thermal cycles [30 s of denaturation at 95 • C, 45 s for annealing at variable temperature (42-44 • C for 16S; 44-50 • C for COI), 1 min for the extension at 72 • C, and final extension for 10 min at 72 • C. The obtained results were observed by 1.5% agarose gel electrophoresis and photographed with Olympus c C-7070 digital camera in a UV M20 UV transilluminator.
The PCR products were purified using the Sureclean purification kit following the manufacturer's protocol. The PCR-purified products were sequenced bidirectionally in automated sequencers (ABI 3100 Genetic Analyzer) at the Department of Technology of the Faculty of Sciences Agricultural and Veterinary Sciences of Jaboticabal, São Paulo State University.
Each genetic sample obtained for the analyses was deposited in the scientific collection of origin (Table S1).

Molecular analyses
A total of 118 sequences used in this study were generated for this project. For the 16S rRNA region, 52 sequences of 518 bp were obtained, of which 29 were A. a. Brazil 1, nine were A. a. Brazil 2, five were A. a. carolinae, five were A. paraguayensis, and four were A. petrunkevitchi. For the COI region, 66 sequences of 589 bp were generated of which 36 were A. a. Brazil 1, 15 were A. a. Brazil 2, six were A. a. carolinae, six were A. paraguayensis, and three were A. petrunkevitchi.
However, an additional 20 sequences (COI) of A. sibogae, A. japonicus, A. serrulatus, and A. indicus, two sequences (16S and COI) of A. petrunkevitchi, and two sequences (16S and COI) of B. faxoni (outgroup) were retrieved from the GenBank database and used for phylogenetic and genetic distance analyses to complement this study (Table S1). As there are COI sequences for more Acetes species than 16S sequences available on GenBank, phylogenetic analysis with the COI gene generated a larger number of clades.
The mean nucleotide composition and genetic distances were estimated using MEGA 5.0 software (Tamura et al., 2011) and the Neighbor-Joining dendrogram based on the Kimura 2-parameter substitution model (Kimura, 1980).
The appropriate models of nucleotide evolution HKY + G for 16S and TPM1uf + G for COI were selected by Bayesian information criterion (BIC) in jModeltest 2.1.4 (Darriba et al., 2012). Selected models and estimated parameters (Table S3) were implemented in the Bayesian inferences and considered for the choice of the closest models in the maximum likelihood analyses. Belzebub faxoni (Borradaile, 1915) (Superfamily Sergestoidea, Family Luciferidae) was included as an outgroup following the most recent global phylogeny (Vereshchaka, Lunina & Olesen, 2016a;Vereshchaka, 2017). Bayesian inference was used to reconstruct the phylogenetic relationships of the species analyzed, with the two genes as distinct partitions in MrBayes v. 3.2.2 (Ronquist et al., 2012). Bayesian inference was performed with 30 million generations in two independent analyses, with five parallel chains each, one ''cold'' and four ''hot''. The parameters were saved every 1,000 simulations. The analysis was completed on attaining stationarity (mean standard deviation <0.01) after the stipulated number of generations. The first quarter of the parameters and trees were discarded as burn-in (Ronquist, Van Der Mark & Huelsenbeck, 2009). The support values of the branches were obtained using the a posteriori probability method.
This analysis was performed for the 16S and COI genes separately and a concatenated matrix of both the trees was generated and edited in the program Figtree v.1.3.1 (Rambaut, 2007).

Morphometric analyses
We used only females (281 individuals) of A. americanus for morphometric analyses. Previous observations focused on the difference in size and shape of the female thelycum between the subspecies, motivating us to obtain potential information to complement our study.
Individuals were sexed based on the presence of petasma (first pleopod) in males and the thelycum (third thoracic sternite) in females (Xiao & Greenwood, 1993).
Morphometric measurements were obtained using a stereo microscope Zeiss c Stemi 2000C connected to an imaging system Zeiss c AxioVision, with an error of up to 0.01 mm. We measured the height (HT) and length (LT) of the thelycum and carapace length (CL).
Following the methodology proposed by Marramà & Kriwet (2017), data were standardized by calculating the ratio between each measure and carapace length (CL), removing the effect of size. The log transformation of data (Log (X+1)) was used to overcome the issue of the non-normal distribution of data by unstretching large-scale values. Moreover, log transformation is useful to considerably reduce variation due to ontogeny (allometric effect) since we assumed that specimens of different developmental stages were subjected to our analyses. A Euclidean distance matrix was constructed using the log-transformed data. Multivariate analysis of variance (PERMANOVA) was applied to test similarities within subspecies and localities (P < 0.005) (Anderson, 2001) using PRIMER software (version 6;Clarke & Gorley, 2006). A Permanova pairwise post-hoc test was performed to further investigate the differences between the subspecies (P < 0.005). Principal component analysis (PCA) was performed to characterize the differences between groups using the measured parameters (HT/CL and LT/CL ratios). Similarity percentage tests (SIMPER) were used to evaluate which measure contributed more to the differentiation between subspecies.

Genetic distance
16S rRNA gene: The intraspecific distances varied from 0% (A. a. USA, A. a. Brazil 2, and A. petrunkevitchi) to 0.20% (A. paraguayensis) ( Table 2). The interspecific distances between congeneric species varied from 0.99 to 11.8%. The distance to the outgroup was 25.9 to 28.5% (Table 3). Regarding the A. americanus subspecies, the lowest interspecific distance was between A. a. Brazil 2 and A. a. USA (0.99%). The highest was A. a. Brazil 1 and A. a. USA (2.26%) ( Table 3).
COI gene: The intraspecific distances varied from 0.02% (A. a. Brazil 1) to 0.97% (A. paraguayensis) ( Table 2). The interspecific distances between the congeneric species varied from 4.86 to 22.3%. The distance to the outgroup was 21.8 to 25.5% (Table 3). Regarding the A. americanus subspecies, the lowest interspecific distance was between A. a. Brazil 1 and A. a. USA (4.86%). The highest was A. a. Brazil 2 and A. a. USA (8.08%) ( Table 3).

Phylogenetic analyses
The 16S rRNA phylogenetic analyses showed the following clades (    The phylogenetic tree constructed based on the concatenated data (16S rRNA and COI) generated the same clades observed in the phylogenetic tree constructed with 16S rRNA and COI separately. In addition, A. a. USA and A. a. Brazil 2 were sister clades (Fig. 5), whereas COI, A. a. USA was a sister clade of A. a. Brazil 1 (Fig. 4).
The phylogenetic trees constructed (16S rRNA, COI, and concatenated data) resulted in the formation of two distinct clades of A. americanus sampled in Brazil with a high support value, in contrast to the clade formed by A. a. carolinae sampled in the United States. Furthermore, the A. americanus group appears to be a sister taxon to all other Acetes (Fig. 4).

Population analyses
The haplotype network exhibited a genetic structure in the three groups, corresponding to those observed in the phylogenetic trees (Fig. 6). Available COI sequences of A. americanus (N = 57) resulted in 12 haplotypes of which six were unique, that is, represented by a single individual. ''Brazil 1 presented the lowest haplotype (h = 0.11) and nucleotide (π = 0.00022) diversities. This group included 34 individuals sharing one haplotype and two individuals with unique haplotypes. ''USA'' and ''Brazil 2'' presented similar high haplotype (h = 0.73 and h = 0.76, respectively) and nucleotide diversities (π = 0.00181 and π = 0.00277, respectively). Analysis of molecular variance (AMOVA) did no detect genetic structuring of any subspecies among the location studied (p > 0.05) ( Table 4).

Morphometric analysis
The ratio between the height and length of the thelycum was determined for several western Atlantic locations (Table 1). The ratio values were higher in A. a. USA and A. a. Brazil 2 than in A. a. Brazil 1 ( Table 1). The studied A. americanus groups (BR1, BR2, and the USA) were morphologically different (P = 0.0002) ( Table 5). Permanova Pairwise tests indicated that at least one of the taxonomic entities is different from the others (P = 0.0001) ( Table 6). However, when comparing A. americanus groups with each other, Pairwise tests indicated that there was a statistically significant difference only between Brazil 1 and Brazil 2 (t = 3.1563; p = 0.0018). The PCA visualization plot shows that A. a. Brazil 1 is more similar to A. a. Brazil 2 than to A. a. USA (Fig. 7). A simplified Simper test showed that the LT/CL RATIO was responsible for the differences between subspecies, contributing more than 73% for Brazil 1, more than 53% for Brazil 2 and more than 61% for the USA (Table 7; Fig. 7).

DISCUSSION
Our analyses revealed the existence of three lineages of A. americanus: A. a. Brazil 1 sensu stricto, A. a. Brazil 2, and A. a. USA. Through molecular analysis, we were also able to identify and contextualize another species of Acetes that occurs in Brazil, A. paraguayensis. Therefore, mitochondrial DNA can be considered an efficient tool for solving taxonomic identification of the genus Acetes at species level.
One of the species concepts widely accepted in systematics (Tsoi, Wang & Chu, 2005) is defined as a group of mating individuals or having the potential for it differing from other groups because they are reproductively isolated (Mayr, 1942). However, there are several alternative concepts of species (De Queiroz, 2007). The haplotype network results suggest that there are three possible lineages for A. americanus: A. a. Brazil 1 sensu stricto,  A. a. Brazil 2, and A. a. USA. Population-based analyses of mitochondrial DNA indicate that entities are reproductively isolated when gene flow is low (Tsoi, Wang & Chu, 2005). The low level of gene flow is essential evidence for speciation (Futuyma, 1998). Therefore, the non-sharing of haplotypes found in this study indicates that A. a. Brazil 1, A. a. Brazil 2, and A. a. USA are genetically distinct, with a low gene flow between them.    also shown genetic homogeneity among populations sampled along the western Atlantic, along the Brazilian coast (Laurenzano, Farias & Schubart, 2012;Terossi & Mantelatto, 2012;Rossi & Mantelatto, 2013;Wieman et al., 2013;Laurenzano, Mantelatto & Schubart, 2013;Carvalho-Batista et al., 2014;Teodoro et al., 2015;Nishikawa, Negri & Mantelatto, 2021). A potential reason for population homogeneity can be ascribed to the high dispersion capacity of planktonic larvae and the absence of barriers to gene flow. Although nothing is known about the larval dispersal of A. americanus, the dispersal power is high for species of the genus Acetes, as they exhibit long planktonic larval stages (∼6 weeks) before becoming juveniles and adults (Rao, 1968). This premise has also been proposed for the other decapods tested (see references above). The levels of genetic divergence (COI) among congeneric species of the crustaceans may vary up to 17%, a high value compared to other animal groups (Costa et al., 2007). Lepidopteran insects have a genetic divergence among congeneric species of only 6.1% (Hebert, Ratnasingham & Waard, 2003). Bird species show a variation of 7.93% (Hebert et al., 2004a), and fish have a 9.93% divergence (Ward et al., 2005). For sergestid shrimp, the rate of genetic divergence (COI) was also high. The genetic divergence found between A. indicus, A. serrulatus, A. japonicus, and A. sibogae ranged from 14.6 to 20.47% (Wong, 2013).
Our results indicated genetic divergence values (COI) from 4.86 to 8.08% between the A. americanus subspecies, which are low when compared with the studies mentioned above. However, if we compare these results to those of studies of cryptic or closely related shrimp species, these values are similar. Species morphologically similar displayed genetic divergences of 2.4 to 7% and were considered different (Gusmão, Lazoski & Solé-Cava, 2000;Lavery et al., 2004;Tsoi, Wang & Chu, 2005;Lagrue et al., 2014). Carvalho-Batista et al. (2019) found higher genetic divergence values (up to 13.5%) among the genus Seabob Xiphopenaeus. However, among some closely related Xiphopenaeus species, the variation ranged from 2.7-3.3%.
Comparing only the measures of the height/length ratio of the female thelycum (Table  1), our results corroborated Omori (1975), which also found that this measurement differed between the two subspecies (A. a. americanus e A. a. carolinae). The values found by Omori (1975) (Table 1). Therefore, if only this character is analyzed, the individual could be misidentified with A. a. USA.
PCA analysis showed a separation of the three subspecies groups (Brazil 1, Brazil 2 and USA) when considering thelycum measurements and the carapace length. In the transition areas where the subspecies occur, however, individuals from Suriname and Puerto Rico identified as A.a. Brazil 1 were close to A. a. USA, and individuals from Mexico identified as A. a. USA were close A. a. Brazil 1 and A. a. Brazil 2. These results point to an interesting geographic pattern, with the separation between individuals collected in the south (Terminos Mexico) and the north of the Gulf of Mexico -GOM (Louisiana). The two portions of the GOM have different water temperatures and have been separated into distinct biogeographic provinces by different authors (Boschi, 2000;Briggs & Bowen, 2012). Additionally, within the GOM, different cyclonic and anti-ciclonic flows occur separating the circulation in each locality (Schmitz Jr et al., 2005) which may be responsible for maintaining the isolation within each one of them. Therefore, this result can be associated with responses to the environmental conditions of the region, as phenotypic variations can be caused by both genetic information and environmental variations (Templeton, 2006). Considering these results, the molecular identification of the subspecies from these transition areas, using the protocols of this study, would be recommended to clarify this issue.
Acetes americanus Brazil 1 is genetically different from A. a. USA. In addition, the specimens sampled in Brazil formed two distinct clades: the first was composed of A. a. Brazil 1 and the second of A. a. Brazil 2. As stated earlier, A. a. Brazil 1 exhibits the diagnostic characteristics of A. americanus americanus, whereas A. a. Brazil 2 exhibits characteristics similar to those of A. amecicanus carolinae. Phylogenetic and population analyses pointed to the divergence of A. a. Brazil 2 from A. a. USA. Usually, a single characteristic to be fixed after reproductive isolation is sufficient for the diagnosis of a species (Mink & Sites Jr, 1996). However, no morphological characteristics that can discriminate these subspecies have been detected thus far. Several studies have shown that many species with few or no morphological characteristics are distinguished by genetic differences (Reuschel, Cuesta & Schubart, 2010;Puillandre et al., 2011;Carvalho, Magalhães & Mantelatto, 2014;Delić et al., 2017;Mandai et al., 2018). A study focusing on the morphological characteristics used to discriminate A. americanus in Brazil should clarify and provide more information about this new possible taxonomic entity. Furthermore, the hypothesis that A. a. Brazil 2 is a different entity, previously described (A. a. louisianensis or A. a. limonensis), cannot be disregarded.

General phylogeny
A previous phylogenetic study of Acetes proposed that the Acetes clade without A. petrunkevitchi (former Peisos petrunkevitchi) never gained robust support, thus considering Peisos as a junior synonym of Acetes (Vereshchaka, Lunina & Olesen, 2016a). Although we carried out an analysis with a robust but limited number of samples, our results confirmed the phylogenetic positioning recovered by both 16S rRNA and COI genes and indicated that A. petrunkevitchi is part of the Acetes group, but in all analyses forming a single clade of ''Acetes paraguayensis + A. petrunkevitchi'', with a high support value for 16S rRNA and concatenated data. As only mitochondrial genes were used, further studies adding nuclear markers should be carried out to test the topology recovered herein.
However, recent studies about the morphology of the male reproductive system and spermatophore of A. petrunkevitchi differed from those of A. americanus, A. marinus and A. paraguayensis, which remains open the discussion about the inclusion of Peisos in the Acetes group (Salti, 2020).
The global morphological phylogeny proposed for the superfamily Sergestoidea showed significant changes in taxonomy, with the description of three new families, particularly because the family Sergestidae was not considered monophyletic (Vereshchaka, 2017). As a result, A. americanus was classified in the new family Acetidae, as proposed by Vereshchaka (2017). The author proposed that a single clade of A. marinus and A. paraguayensis within Acetidae received high bootstrap support. Our concatenated molecular phylogeny indicated a close relationship between A. paraguayensis and A. petrunkevitchi in a separate clade, as mentioned above. Additional samples of these species, including A. marinus, confirm this hypothesis. This molecular reconstruction sheds light on the unsolved evolutionary relations between the species of the genus Acetes, which should be investigated using more comprehensive integrated studies and the addition of nuclear markers.
• Sarah de Souza Alves Teodoro performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.
• Fernando L. Mantelatto conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, grant support coordinator, and approved the final draft.

Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers): All specimens used in our analysis were collected under Federal autorization from the Instituto Chico Mendes de Biodiversidade/ICMBio (#11777-2, #23008-1).

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences: The group of mitochondrial (16S and COI) sequences are available at GenBank: OP035650-OP035701 (16S) and OP060465-OP060530 (COI).